1a. Objectives (from AD-416)
Provide toxicological data and analytical methodology for microbial toxins that will help ensure a safe food supply. (1) Develop new assays for bacterial toxins and their variants, using immunological and other methods, with emphasis on applicability to practical problems facing the food industry and regulatory agencies. Develop new monoclonal antibody (mAb)-based assays for botulinum neurotoxins (BoNTs), non-toxic neurotoxin-associated proteins, and Shiga toxins (Stx), and optimize antibodies for biosensor applications. Develop methodology for detection of Shiga toxin-producing E. coli (STEC) and a multiplex bead-array assay for detecting Stx and STEC pathogenicity/virulence factors. Develop improved activity assay for staphylococcal enterotoxins. (2) Calibrate in vitro methodology against established animal bioassays, and develop new data on the bioavailability of toxins, the impact of food processing on toxin activities, and the significance of antibody-mediated clearance on toxicity, especially via the oral route of intoxication. Determine the bioavailability of different botulinum neurotoxin serotypes. Validate new toxin assays using activity assays.
1b. Approach (from AD-416)
For the first objective, the general approaches are to exploit immunoassays, especially enzyme-linked immunosorbent assay (ELISA), immuno-polymerase chain reaction (iPCR), and bead array assays because of their versatility, robustness, and sensitivity; and to develop activity assays. The mAbs developed for immunoassay will also have important utility for sample preparation and potential for diagnostic/therapeutic applications. Development of new toxin-specific mAbs will exploit a variety of immunogens, including toxoids and recombinant polypeptide chains corresponding to different domains of the toxin chains. Methodologies to optimize antibodies include the use of flow cytometry to test and select hybridoma cell lines. Structurally different antibodies such as IgY and single-chain antibodies will also be developed and compared to mAbs. Optimal capture/detection antibody pairs will be identified using ELISA and assay performance will be investigated with respect to robustness. Selected capture antibodies will be coupled to immunomagnetic beads for use in sample preparation. Assays will be evaluated in the food matrices of principal interest: milk, juices, liquid eggs, and ground meat and poultry products. For samples that produce a high background signal, matrix interference, or poor recovery, simple preparative methods will be tested, such as differential centrifugation, filtration, or immunomagnetic bead separations. Similar methodology will be used to develop antibodies and assays for accessory proteins found in toxin preparations as secreted by bacteria. Activity assays report active toxin. They will be especially useful to measure toxins in the presence of thermally inactivated and degraded proteins that are found in processed food samples. Assays that measure the activity of selected toxins (such as Stx and staphyloccocal enterotoxins) will utilize existing and new cell lines that are sensitive to active toxin. Suitable readout systems include cell lines that produce reporter molecules in response to toxin. For the second objective, the general approach is to relate variations in toxin structure to toxicity, bioavailability, and responses in detection systems. Bioassays and cell-based assays will be used to assess the impact of food processing on toxin activity and bioavailability. Dose-response and bioavailability will be determined for BoNT holotoxins and toxin complexes. We will determine the effect of accessory proteins on toxicity. The transit time for passage through the intestinal epithelium will be determined for holotoxin and toxin complexes in a model system, polarized colonic epithelial translocation assay. The protective effect of newly developed antibodies will be determined for various BoNTs. Assay validation is based on side-by-side comparison of samples in different assay systems. Active toxin concentration will be estimated by biochemical and cellular assays. Bioassays will be used for assessment of toxicity of unknown concentrations of toxins for comparison with in vitro assays, especially for toxin in raw and processed food matrices. Replacing 5325-42000-043-00D (2/11).
3. Progress Report
This project replaced #5325-42000—043-00D on 28 December 2010 and emphasizes bacterial toxins. High-affinity monoclonal antibodies (mAbs) were developed for sensitive detection of botulinum neurotoxins (BoNTs) in enzyme-linked immunosorbent assays (ELISA). New immunochemical methods were developed using chemiluminescence, electrochemiluminescence (ECL) and polymerase chain reaction (PCR) technologies to improve detection sensitivity in complex biological and environmental matrices (water, sera, feces, and foods such as ground beef). Shiga toxins (Stx) are toxic glycoproteins belonging to the type II group of ribosome inactivating proteins. We developed assays for these toxins using immuno-PCR technology that combines the advantages of flexible and robust immunoassays with the exponential signal amplification of PCR. We were able to detect 10 picograms/ml of Stx in environmental samples and validation in food matrices was begun. Shiga toxin-producing Escherichia coli (STEC) cause disease by producing Shiga toxins 1 and 2 (Stx1 and 2) and other virulence factors. Few tools are available to food safety and health professionals for identification of non-O157 STEC. We have developed a test that detects the ten most common STEC O-serotypes. Our test uses magnetic fluorescent microbeads in which each serotype is detected by a different color fluorescent bead, to which specific mAbs are coupled to provide specific binding activity. Assay performance was tested in multiple laboratories using 11 different STEC serotypes. Samples of lettuce and milk spiked with very low levels of E. coli were correctly identified with no false positives or false negatives. This microbead-based immunoassay could be extended to detect additional foodborne pathogens and their toxic markers simultaneously. Understanding the toxicokinetics and pathological effects of toxins following oral intoxication is valuable for advancing food safety and defense and has been facilitated by the newly developed assay technologies. Monoclonal antibodies against BoNT/A and BoNT/B were optimized for the detection of toxin in sera using a multiwell plate-based ECL assay system. In mouse model systems of intoxication, the ECL assay system was used to study the toxicokinetics of BoNTs. An atoxic form of BoNT/A was developed to allow detailed studies of the movement of the toxin in exposed animals. Other studies in our laboratory helped to elucidate a novel mechanism by which staphylococcal enterotoxins produced by the bacterium Staphylococcus aureus cause food poisoning and contribute to several human diseases. A better understanding of the etiology of intoxication will help in the design of more effective diagnostic and therapeutic methods.
1. Microbead assay for Shiga toxins and toxin-producing E. coli. Food safety regulators and industrial partners lack a simple test for simultaneous detection of E. coli O157:H7 as well as the toxins produced by O157 and other disease-causing E. coli. A rapid, sensitive assay was developed for detecting Shiga toxin-producing E. coli (STEC) and the major toxins produced by STEC, Shiga toxin 1 (Stx1) and 2 (Stx2). The test works with magnetic fluorescent microbeads capable of binding to STEC and the toxins, while simplifying recovery of the bacteria from food. The specificity and sensitivity of this immunoassay were tested against a collection of STEC isolates belonging to various serotypes. All the samples were correctly identified with no false positives or false negatives. Because of its improved speed, the test is ideal for use in food processing plants to allow in-house testing of products in less than 24 hours. The test will directly benefit regulatory agencies by minimizing analysis time and enabling the recovery of bacteria, necessary before contaminated food can be recalled.
2. Windows of opportunity for neutralization of botulinum neurotoxin. Botulinum neurotoxins (BoNTs), responsible for botulism food poisoning, are rapidly absorbed in small amounts that they are very difficult to detect, even though they can be very dangerous. Scientists in Albany, CA, developed monoclonal antibodies specific for BoNTs and tested them for their ability to provide protection against botulism exposure in a mouse model system. Following intravenous and oral exposures to lethal levels of toxin, the timing of antibody neutralization of the toxin was determined. The results provided new information on the toxicity of BoNTs and revealed windows of opportunity for therapeutic treatment with antibody. A better understanding of the biology of toxins in animals and the factors that affect their toxicity, is a valuable tool for advancing food safety and defense.
3. Ultra-sensitive test for Shiga toxin 2. Although E. coli disease can be caused by the presence of even a few organisms, existing methods for detection of the bacteria require many millions of bacteria, typically obtained through overnight cultural enrichment. ARS scientists in Albany, CA, developed an immuno-polymerase chain reaction (IPCR) assay for the detection of Shiga toxin 2 (Stx2), which is produced by disease-causing E. coli. Application of the IPCR assay to 23 non-enriched samples collected from the environment revealed that the IPCR detected Stx2 in all 15 samples that were shown to be STEC-positive by established culture methods. This assay provides an extremely rapid, perfectly sensitive, and specific new method for detecting pathogenic E. coli in food and environmental samples.